Sodium coupled, carrier mediated transport of bile acids cannot be demonstrated in the ileum of the neonate, but is expressed prior to weaning in several species of mammals. Therefore, the overall objective of this proposal is to use cellular and molecular biological approaches to define the mechanisms for the ontogenic expression and regulation of the ileal-brush border membrane transport system for bile acids. The ileal bile acid transport protein, identified by its developmental appearance and isolated by affinity chromatography, will be used to generate polyclonal and monoclonal antibodies. Specific antibodies against the carrier protein will be used to inhibit bile acid transport by brush border plasma membrane vesicles and to follow the ontogenic expression of the carrier protein on the plasma membrane by immunochemical quantitation and immunofluorescence microscopy. Monoclonal antibodies will be used to probe the structure of the transporter (including binding sites for substrates and inhibitors of the carrier). To confirm that the membrane protein is actually involved in sodium-dependent bile acid transport and is sufficient to mediate this transport process, we will employ a solubilized total membrane fraction or a fraction purified with the affinity column to reconstitute transport activity into proteoliposomes. A cDNA probe encoding the ileal brush border bile acid transport protein will be cloned using several complimentary strategies. The authenticity of an identified cDNA will be verified through assaying of Xenopus oocytes for bile acid transport after injection of the oocytes with the RNA fraction of interest. Hydropathy analysis of the amino acid sequence, deduced from the cDNA clone, will provide insight into the functional organization of the transport protein within the plasma membrane. The cDNA probe for the bile acid transporter will be used in slot blot and northern blot assays to determine if ontogenic regulation of transport activity occurs by transcriptional or translational mechanisms or whether this transporter is expressed in the colon during development. Expression of the carrier protein and message levels will also be assayed after corticosteroid injection of suckling rats or in response to feeding of exogenous bile acids. The cDNA probe will be utilized for in situ hybridization studies to determine at what location on the crypt-villus axis the transport protein is first expressed. New insight into the mechanisms and regulation of intestinal bile acid transport will ultimately be of value in understanding alterations in absorptive function which occur during ileal disease and during perinatal development.